Association Between Gut Regulatory Hormones and Post-operative Weight Loss Following Gastrectomy in Patients With Gastric Cancer

Background/Aims@#Post-operative weight loss in patients with gastric cancer lead to a poor quality of life and long-term survival. This study aims to evaluate the effects of gut regulatory hormones on post-operative weight loss in patients with subtotal gastrectomy for gastric cancer. @*Methods@#This prospective study was conducted for 12 months post-surgery in 14 controls and 13 gastrectomy patients who underwent subtotal gastrectomy for gastric cancer. Serum plasma ghrelin, glucagon-like peptide-1, gastric inhibitory peptide-1, peptide YY, insulin, and homeostatic model assessment for insulin resistance responses to a standardized test meal were recorded at multiple time points before and after gastrectomy at 4 and 12 months. @*Results@#The mean weight difference between the pre-operative state and the 4-month period was significantly reduced to 6.6 kg (P = 0.032), but significant weight reduction was not observed from 4 months to 12 months. The plasma levels of glucagon-like peptide-1, gastric inhibitory peptide-1, and peptide YY were significantly increased 4 months postoperatively compared to the pre-operative state (all P= 0.035); however, pre-operative levels and relative changes over a period of 0-4 months of hormones were not correlated with body weight changes. Only the pre-operative ghrelin at peak had a negative correlation with changes in weight reduction in the 4 months after surgery (P = −0.8, P = 0.024). @*Conclusions@#Significant weight reduction was common after subtotal gastrectomy for gastric cancer with a negative correlation pre-operative plasma ghrelin levels. Incretin hormones are modestly but significantly increased after subtotal gastrectomy; however, these changes did not affect the weight changes.

Hyperoxygenation Ameliorates Stress-induced Neuronal and Behavioral Deficits

Hyperoxygenation therapy remediates neuronal injury and improves cognitive function in various animal models. In the present study, the optimal conditions for hyperoxygenation treatment of stress-induced maladaptive changes were investigated. Mice exposed to chronic restraint stress (CRST) produce persistent adaptive changes in genomic responses and exhibit depressive-like behaviors. Hyperoxygenation treatment with 100% O2 (HO2 ) at 2.0 atmospheres absolute (ATA) for 1 h daily for 14 days in CRST mice produces an antidepressive effect similar to that of the antidepressant imipramine. In contrast, HO2 treatment at 2.0 ATA for 1 h daily for shorter duration (3, 5, or 7 days), HO2 treatment at 1.5 ATA for 1 h daily for 14 days, or hyperbaric air treatment at 2.0 ATA (42% O2 ) for 1 h daily for 14 days is ineffective or less effective, indicating that repeated sufficient hyperoxygenation conditions are required to reverse stress-induced maladaptive changes. HO2 treatment at 2.0 ATA for 14 days restores stress-induced reductions in levels of mitochondrial copy number, stress-induced attenuation of synaptophysin-stained density of axon terminals and MAP-2-staining dendritic processes of pyramidal neurons in the hippocampus, and stress-induced reduced hippocampal neurogenesis. These results suggest that HO2 treatment at 2.0 ATA for 14 days is effective to ameliorate stress-induced neuronal and behavioral deficits.

Isolation and Characterization of Vaccine Candidate Genes Including CSP and MSP1 in Plasmodium yoelii

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.

Rapid Assessment of Microbiota Changes in Individuals with Autism Spectrum Disorder Using Bacteria-derived Membrane Vesicles in Urine

Individuals with autism spectrum disorder (ASD) have altered gut microbiota, which appears to regulate ASD symptoms via gut microbiota-brain interactions. Rapid assessment of gut microbiota profiles in ASD individuals in varying physiological contexts is important to understanding the role of the microbiota in regulating ASD symptoms. Microbiomes secrete extracellular membrane vesicles (EVs) to communicate with host cells and secreted EVs are widely distributed throughout the body including the blood and urine. In the present study, we investigated whether bacteria-derived EVs in urine are useful for the metagenome analysis of microbiota in ASD individuals. To address this, bacterial DNA was isolated from bacteria-derived EVs in the urine of ASD individuals. Subsequent metagenome analysis indicated markedly altered microbiota profiles at the levels of the phylum, class, order, family, and genus in ASD individuals relative to control subjects. Microbiota identified from urine EVs included gut microbiota reported in previous studies and their up- and down-regulation in ASD individuals were partially consistent with microbiota profiles previously assessed from ASD fecal samples. However, overall microbiota profiles identified in the present study represented a distinctive microbiota landscape for ASD. Particularly, the occupancy of g_Pseudomonas, g_Sphingomonas, g_Agrobacterium, g_Achromobacter, and g_Roseateles decreased in ASD, whereas g_Streptococcus, g_Akkermansia, g_Rhodococcus, and g_Halomonas increased. These results demonstrate distinctively altered gut microbiota profiles in ASD, and validate the utilization of urine EVs for the rapid assessment of microbiota in ASD.

Phenotypic and Functional Analysis of HL-60 Cells Used in Opsonophagocytic-Killing Assay for Streptococcus pneumoniae

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

Phenotypic and Functional Analysis of HL-60 Cells Used in Opsonophagocytic-Killing Assay for Streptococcus pneumoniae

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

Hepatocyte Growth Factor is the Key Cytokine in Stimulating Potential Stem Cells in the Cord Blood into Hepatic Lineage Cells

BACKGROUND: This study was designed to investigate the role of the hepatocyte growth factor (HGF) with regards to differentiation of somatic stem cells originating from the human umbilical cord blood (UCB) into hepatic lineage cells in vitro culture system. METHODS: Mononuclear cells from UCB were cultured with and without HGF based on the fibroblast growth factor (FGF)-1, FGF-2, and stem cell factor. The cultured cells were confirmed by immunofluorescent staining analysis with albumin (ALB), cytokeratin-19 (CK-19), and proliferating cell nuclear antigen (PCNA) MoAb. ALB and CK-18 mRNA were also evaluated by reverse transcription-polymerase chain reaction. In order to observe changes in proliferating capacity with respect to the cultured period, CFSE with affinity to proliferating cells were tagged and later underwent flow cytometry. RESULTS: In the HGF-treated group, cultured cells had a large oval shaped appearance with adherent, but easily detachable characteristics. In the HGF-non treated group, these cells were spindle-shaped with strong adherent characteristics. Expressions of ALB and CK-19 were evident in HGF-treated group compared to non-expression of those in to HGF-non treated group. Dual immunostaining analysis of the ALB producing cells showed presence of PCNA in their nuclei, and ALB and CK-18 mRNA were detected on the 21st day of cultured cells in the HGF-treated group. CONCLUSION: Our findings suggest that HGF has a pivotal role in differentiating somatic stem cells of human UCB into hepatic lineage cells in vitro.

Plasmodium falciparum Cultivation Using the Petri Dish: Revisiting the Effect of the 'Age' of Erythrocytes and the Interval of Medium Change

Differences in the characteristics of the culture conditions can influence the multiplication rate of Plasmodium falciparum. The Petri dish method is one of the most popular methods of cultivating this parasite. In many previous studies, ideal culture conditions of the Petri dish method were achieved by using erythrocytes collected from blood that had been stored for at least 2 weeks, with daily changes of the medium. In the present study, we studied the multiplication rate of P. falciparum in cultures containing erythrocytes of various ages together with changing the medium at various intervals of time. Our results strongly suggest that the rate of in vitro multiplication of P. falciparum was higher in freshly collected erythrocytes than in aged erythrocytes regardless of the anticoagulant and that when the parasitemia is lower than 8% with a hematocrit of 5%, the medium change interval can be as long as 48 hr without a great reduction in the rate of multiplication.

Investigation of Chemotactic Activities in Differentiated HL-60 Cells by a Time-lapse Videomicroscopic Assay

BACKGROUND: Chemotaxis is one of the cardinal functions of leukocytes, which enables them to be recruited efficiently to the right place at the right time. Analyzing chemotactic activities is important not only for the study on leukocyte migration but also for many other applications including development of new drugs interfering with the chemotactic process. However, there are many technical limitations in the conventional in vitro chemotaxis assays. Here we applied a new optical assay to investigate chemotactic activities induced in differentiated HL-60 cells. METHODS: HL-60 cells were stimulated with 0.8% dimethylformamide (DMF) for 4 days. The cells were analyzed for morphology, flow cytometry as well as chemotactic activities by a time-lapse videomicroscopic assay using a chemotactic microchamber bearing a fibronectin-coated cover slip and an etched silicon chip. RESULTS: Videomicroscopic observation of the real cellular motions in a stable concentration gradient of chemokines demonstrated that HL-60 cells showed chemotaxis to inflammatory chemokines (CCL3, CCL5 and CXCL8) and also a homeostatic chemokine (CXCL12) after DFM-induced differentiation to granulocytic cells. The cells moved randomly at a speed of 6.99+/-1.24 micrometer/min (n=100) in the absence of chemokine. Chemokine stimulation induced directional migration of differentiated HL-60 cells, while they still wandered very much and significantly increased the moving speeds. CONCLUSION: The locomotive patterns of DMF-stimulated HL-60 cells can be analyzed in detail throughout the course of chemotaxis by the use of a time-lapse videomicroscopic assay. DMF-stimulated HL-60 cells may provide a convenient in vitro model for chemotactic studies of neutrophils.

Myeloablative Treatment Supported by Autologous Stem Cell Infusion with Neuroblastoma

Bcr-abl antisense oligodeoxynucleotides (AS-ODNs) have provided evidence of an antileukemia effect when tested in vitro against Philadelphia-positive cells. In order to investigate the efficacy of AS-ODNs as purging agents in chronic myeloid leukemia (CML) patients, K562 cells, a human CML cell line, were treated in vitro with various types of AS-ODNs and interferon-alpha. Cells were treated in vitro for 0 and 36 hr with 40 microgram/mL of AS-ODNs, respectively, and incubated at 37 degrees C for 36 hr. Cytotoxic effects were measured by counting the number of viable cells as well as by MTT test. Clonogenic activities were evaluated by methylcellulose culture for 2 weeks. The effects of purging agents on the rearrangement of bcrabl gene were evaluated by RT-PCR. AS-ODNs inhibited the proliferation of K562 cells with time in cell count assay and MTT test. AS-ODNs were superior to INF-alpha in inhibiting clonogenic activity (recovery rate; 26.3% vs 64.0%). After incubation with bcr-abl AS-ODNs primers and mRNA isolated from K562 cells, positive bands were abolished, especially of b3a2 type and phosphorothioate type. Our results suggest that AS-ODNs mediated purging may be one of the efficient methods and that autograft may be an alternative treatment for allograft in high-risk group patients of CML if they do not have a stem cell donor.

Circulating Cytokine Levels and Changes During the Treatment in Patients with Active Tuberculosis in Korea / 결핵

BACKGROUND: The cell-mediated immune reaction to tuberculosis infection involves a complex network of cytokines. The extent of inflammation, tissue damage and severity of the disease suggested to be determined by the balance between extent and duration of the proinflammatory cytokine response versus those of the suppressive cytokines. The systemic cytokine response in pathogenesis of tuberculosis can be assessed by measuring serum cytokine levels. METHOD: Serum interleukin-1 beta(IL-1 ), IL-2, IL-4, IL-6, IL-10, IL-12(p40), tumor necrosis factor-alpha(TNF-alpha), interferon-gamma(IFN-gamma) and transforming growth factor-beta(TGF-beta) levels were measured in 83 patients with pulmonary tuberculosis, 10 patients with endobronchial tuberculosis before treatment and 20 healthy subjects by using a sandwich ELISA. In patients with pulmonary tuberculosis, they were divided into mild, moderate and far advanced group according to the severity by ATS guidelines. To compare with those of pretreatment levels, we measured serum IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12(p40), TNF-alpha, IFN-gamma and TGF-beta levels in 45 of 83 patients with pulmonary tuberculosis after 2 and 6 months of treatment. RESULTS: 1) In sera of patients with active pulmonary tuberculosis(n=83), IL-1beta, IL-6(p<0.05), TNF-alpha, and IFN-gamma were elevated and TGF-beta was decreased comparing to control. IL-2, IL-12(p40), IL-4 and IL-10 were similar between the patients with tuberculosis and control. 2) In endobronchial tuberculosis, IL-6 and TNF-alpha were elevated and TGF-beta was decreased comparing to control. IL-12(p40) seemed to be elevated comparing to pulmonary tuberculosis. 3) Far advanced tuberculosis showed markedly elevated IL-6 and IFN-gamma level(p<0.05). 4) The significant correlations were noted between IL-1, IL-6 and TNF-alpha and between IL-12, IL-2 and IL-4(p<0.01). 5) After 2 and 6 months of standard treatment, the level of IL-6 and IFN-gamma was significantly decreased(p<0.05). CONCLUSION: These results showed that an altered balance between cytokines is likely to be involved in the extent of inflammation, tissue damage and severity of the disease tuberculosis. But, it should be considered diversities of cytokine response according to type of tuberculosis and immunity in clinical application and interpreting future studies.

Antitumor Effects of Arsenic Trioxide on Neuroblastoma / 대한소아혈액종양학회지

PURPOSE: We aimed to study the feasibility of arsenic trioxide as a treatment of neuroblastoma which has the ability to differentiate into nonmalignant cells like acute promyelocytic leukemia. METHODS: To determine the effects of arsenic trioxide in various concentrations and exposure time on the survivial of neuroblastoma cell lines, SH-SY5Y and SK-N-AS cells were cultured in RPMI 1640 media with 1 to 20muM concentration of arsenic trioxide. Apoptosis was measured with flow cytometry by staining with 7-aminoactinomycin D. Cell cycle was assessed by monitoring the DNA contents by flow cytometry. Arsenic trioxide induced cell morphologic changes were also observed with May-Grunwald-Giemsa stain under a light microscope. RESULTS: Arsenic trioxide induced apoptosis in SH-SY5Y cells earlier in the same concentration and to a more severe degree with the same exposure time than in HL-60 cells. The apoptosis induced by arsenic trioxide was steeply increased to 79.3 10.1% at 24 hours and then maintained a plateau on 20muM concentration, while increasing steadily to 40.2 6.5% until 72 hours on 5muM concentration. The proliferating cell proportion in S/G2/M phase was decreased with arsenic trioxide concentration and with exposure time in both SH-SY5Y and HL-60 cells, especially more so with the SH-SY5Y cells. The cellularity was decreased and more apoptotic cells could be observed in the arsenic trioxide treatment group than in untreated control group. CONCLUSION: As in acute promyelocytic leukemic cells, arsenic trioxide induced apoptosis and cell cycle arrest of proliferating phase in neuroblastoma cells.

Differentiation and Apoptosis of the Erythroid, Myeloid and Megakaryocytic Lineage Cells in vitro Culture of Cord Blood CD34+ Cells Using Various Cytokines / 대한혈액학회지

BACKGROUND: In vitro generated cells from cord blood (CB) CD34+ cells increase cell dose and may reduce the severity and the duration of pancytopenia after transplantation. But safe engraftment for adolescents and adults is still not predictable and standardized. We attempted to establish a clinically application of in vitro generated cells by investigating the use of cytokines for the culture of CB CD34+ cells. METHODS: CD34+ cells, purified from four separate human CB units by magnetic bead selection, were cultured in IMDM with several cytokines. Differentiation of the in vitro generated cells has been confirmed by flowcytometry with specific erythroid, myeloid and megakaryocytic lineage surface markers. And apoptosis of these cells also was analysed with annexin V staining and morphologic analysis under the electron microscopic examination was done, simultaneously. RESULTS: Purified CB CD34+ cells were expanded with differentiation from CD38- to CD38+ expression with time. CB CD34+ cells could be terminally differentiated into erythroid (GPA+) and myeloid (CD33+/CD15+) lineage cells without apoptosis (annexin-V - ) in the presence of EPO and G-CSF, respectively. Megakaryocytic differentiation was partially arrested in early stage due to apoptosis in the presence of TPO. Morphological examination using electron microscope revealed all stages of erythroid and myeloid lineage cells without apoptosis, and apoptotic megakaryocytic lineage cells of early stage. But we could observe the small number of fully maturated platelets. CONCLUSION: CB CD34+ cells were terminally differentiated to erythroid, myeloid, and megakaryocytic lineage cells with or without apoptosis by several cytokines.

The Prognostic Significance of the Overexpression of HER-2/ neu in Korean Gastric Carcinomas and the In Vitro Effects of Anti-HER-2/neu Antibody on Cell Growth in the Gastric Carcinoma Cell Lines / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The HER2 gene encodes a 185-kd transmembrane glycoprotein receptor (p185(HER2)) that has partial homology with the epidermal growth factor receptor (EGFR) and shares intrinsic tyrosine kinase activity. The HER2 gene has been found to be amplified in various human cancers and to be associated with poor prognosis. The authors investigated the correlation between clinicopathologic factors and the overexpression of the p185(HER2) in Korean gastric adenocarcinoma patients, and determined whether the antiproliferative effects of anti- p185(HER2) antibody can also be observed on gastric cancer cell lines that overexpress this growth factor receptor. MATERIALS AND METHODS: We evaluated the relationship between p185(HER2) overexpression and clinicopathological features in 94 (M: F=52: 42) gastric adenocarcinoma patients (median age 59 years). Protein expression was analysed by immunohistochemical staining in paraffin embedded tissues with monoclonal antibody for p185(HER2). To explore the role of humanized anti-p185(HER2) monoclonal antibody trastuzumab (Herceptin ) in vitro, the growth curve of Korean gastric cancer cells that overexpress the p185(HER2) protein was studied and a cell cycle analysis was performed. RESULTS: p185(HER2) overexpression correlates positively with lymph node metastasis (p=0.002), distant metastasis (p=0.01), AJCC classification (p=0.01), higher relapse rate p=0.001), and a tendential association with the pT stage (p=0.054). p185(HER2) overexpression was found to be more frequent in advanced gastric cancer than early gastric cancer (54.1% vs 24.2%, p=0.008). Patients with overexpression of p185(HER2) were found to have significantly lower relapse-free (p=0.003) and overall survival (p= 0.0004) than patients without overexpression. Among several Korean gastric cancer cell lines, SNU-1, SNU-5, and SNU-620 overexpress p185(HER2). Trastuzumab inhibited the proliferation of p185(HER2) overexpressed Korean gastric cancer cell line by 21% with down-regulation of p185(HER2) protein expression. DNA fluorescence flow cytometry of propidium iodide-stained nuclei showed a reduction in the fraction of the S phase following treatment with trastuzumab. CONCLUSIONS: Taken together, our observations suggest the potential prognostic significance of p185(HER2) overexpression in Korean gastric adenocarcinoma patients and point to the need for further research on this mechanism. This suggests the possible use of p185(HER2) as a therapeutic target in gastric cancer.

Soluble IL-2R, IFN-gamma and Neopterin as Immunologic Markers in Patients with Tuberculosis / 결핵

BACKGROUND: The cell-mediated immune response plays an important role in tuberculosis. After being activated by mycobacterial antigens, T lymphocytes express a high affinity receptor (IL-2R) for interleukin-2 (IL-2) on their own surface and release a soluble fraction of the IL-2 receptor (sIL-2R) from the cell membrane into the circulation. Neopterin is a metabolite of guanosine-triphosphate, which is produced by stimulated macrophages under the influence of IFN-gamma with a T lymphocyte origin. Therefore, the utility of sIL-2R, IFN-gamma and the neopterin levels as immunologic indices of the cell-mediated immune response and severity of disease in patients with pulmonary tuberculosis was assessed. METHOD: The serum sIL-2R, IFN-gamma and neopterin levels were measured in 39 patients with pulmonary tuberculosis, 6 patients with tuberculous lymphadenitis prior to treatment and 10 healthy subjects. The serum and pleural sIL-2R, neopterin and ADA levels were measured in 22 patients with tuberculous pleurisy. The patients with pulmonary tuberculosis were divided into a mild, moderate and severe group according to the severity by ATS guidelines. To compare the results from these patients with those of the pretreatment levels, the sIL-2R, IFN-gamma and neopterin levels were measured in 36 of the 39 patients(1 patient, expired; 2 patients were referred to a sanitarium) with pulmonary tuberculosis after 2 months of treatment. RESULTS: 1) The serum sIL-2R and IFN-gamma levels were elevated in patients with tuberculosis when compared to those of healthy subjects (0.05). The neopterin concentration in the serum was significantly lower in patients with pulmonary tuberculosis(2967+/-2132.8 pg/ml) than in healthy controls(4949+/-1242.1 pg/ml)(p0.05), 41 52.8 pg/ml to 22+/-23.9 pg/ml(p<0.05), respectively, after 2 month of treatment. The mean serum neopterin levels increased from 3158+/-2272.6 pg/ml to 3737+/-2307.5 pg/ml(0.05) after a 2 month of treatment. These findings were remarkable in the severe group of pulmonary tuberculosis with a clinical correlation. 4) In the patients with tuberculous pleurisy, the serum sIL-2R and ADA were significantly higher than those in the pleural fluid, However, the neopterin levels in the sera and pleural effusion were similar. CONCLUSION: On the basis of this study, sIL-2R, IFN-gamma and neopterin measurements may not only provide an insight into the present state of the cell-mediated immune response, but also serve as parameters monitoring of the prognosis of the disease, particularly in patients with severe pulmonary tuberculosis. In addition, an assay of the pleural sIL-2R levels might signal a stimulated local immunity including T cell activation in the tuberculous pleural effusion.

A Study of Receptor Expression in Leukocytes by Quantitative Fluorescence Measurement

PURPOSE: It is well known that the receptors for Fcgamma moiety of IgG(FcgammaR) are instrumental in antibody-mediated clearance of microorganisms by granulocytes and monocytes, and immune regulation in lymphocytes. Furthermore, complement receptor-3(CR3) is also important in neutrophilic adhesion, diapedesis, and phagocytic functions. In an attempt to examine their roles in neonates, we compared the expressions of FcgammaRs and CR3 in cord blood leukocytes with those expressed in leukocytes in samples of blood obtained from adult subjects. METHODS: Cord blood was obtained from 30 full-term newborn and peripheral blood from 30 adult volunteers. In both groups, we measured three Fcgamma receptors and one adhesion molecule, CR3 on granulocytes, monocytes and lymphocytes using a whole blood method with flow cytometry and quantitative bead standards to enumerate the cell surface receptors. RESULTS:Compared to those observed in adult blood, the proportions of FcgammaRI+ granulocytes were significantly higher in cord blood. By contrast, the proportions of FcgammaRII+ or FcgammaRIII+ granulocytes and the number of FcgammaRIII were significantly lower in cord blood. The proportions of FcgammaRI+, FcgammaRII+, and CD18+ bearing monocytes and the number of FcgammaRII in lymphocytes were also significantly lower in cord blood as compared to adult blood. CONCLUSION: There were differences in the proportions of cells expressing FcgammaRs and CR3 and in the number of FcgammaRs and CR3 in granulocytes, monocytes, and lymphocytes between cord blood and peripheral blood from adult subjects. These may contribute to the difference in immune capability that is known to exist between neonates and adult subjects.

Analysis of stromal Cells Developed from Cord Blood CD34+ Cells

BACKGROUND: Cytokine-mediated ex vivo expansion has been proposed as a means of increasing the number of cord blood (CB) hematopoietic stem cells for transplantation. As well as stem cell number, stromal cells are necessary for functional maturation of hematopoiesis. The purpose of this study was to analyze the development of stromal cells during ex vivo expansion of CB CD34+ cells. METHODS: CD34+ cells were purified from CB by magnetic bead selection. The levels of of interleukin-3, interleukin-1 beta, interleukin-6, granulocyte macrophage-colony stimulating factor and tumor necrosis factor-alpha were measured in culture supernatants on 0, 1, 2, and 3 weeks, using ELISA techniques. CB CD34+ cells were expanded in Iscoves modified Dulbeccos medium in the presence of several cytokines. The expression of E-selectin, vascular cell adhesion molecule- 1, intercellular adhesion molecule- 1, platelet/endothelial cell adhesion molecule-1, von Willebrand factor, vimentin, and CD14 in newly developed stromal cells was examined by immunocytochemical method. Relevant extracellular matrix (ECM) proteins and proper cytokines were also assayed for the most suitable condition for expansion of stromal cells. RESULTS: Several cytokines were found to have been produced by CB CD34+ cells as well as bone marrow-derived CD34+ cells. During ex vivo expansion of CB CD34+ cells, stromal cells appeared in the culture by day 4 and expanded over the following 7- 10 days before being confluent by day 2 1. These cells expressed surface markers characteristic of cells of endothelial lineage. Furthermore, these stroaml cells also expanded effectively when treated with thrombopoietin+flt-3 ligand+stem cell factor+leukemia inhibitory factor or 0. 1% poly-L-lysine-coated wells. CONCLUSION: Stromal cells were developed during ex vivo expansion of CB CD34+ cells and that this development could be enhanced further by treating the stromal cells with cytokines or ECM.

Cell cycling status of human cord blood CD34+ cells during ex vivo expansion is related to the level of very late antigen expression

Very late antigen-4 (VLA-4), which binds to the extracellular matrix protein fibronectin, is an integrin molecule known to be modulated during mobilization of CD34+ cells, and to be involved in signaling the mobilization stimuli. On the hypothesis that cell cycling status might be different depending on the level of VLA-4 expression, we investigated the DNA contents of human cord blood CD34+ cells during ex vivo expansion by recombinant human thrombopoietin and flt3-ligand with simultaneous measurement of surface VLA-4 at the 1st and 4th week. During this ex vivo expansion, expression of VLA-4 increased and almost all cells became VLA-4+ until the 4th day of culture. Expression of VLA-4 was maintained in the major population of the cultured cells until the 4th week. The cells in S/G2/M phase were greater in number in VLA-4 high fraction than in VLA-4 low fraction (n=4, p<.001). Furthermore, the fraction of cells in S/G2/M phase increased as the expression of VLA-4 became higher. These results suggest that cord blood CD34+ cells expressing high levels of VLA-4 have more proliferative activities.

The Effect of rhG-CSF and rhGM-CSF on Expression of Fc gamma Receptors and beta2 Integrin and Respiratory Burst Function in Human Granulocytes

PURPOSE: Receptors for the Fc of IgG(FcvR) and a beta2 integrin molecule, CD11c/CD18 are important in clearance of microbes by granulocytes. We performed an in vitro study on the effect of recombinant human granulocyte colony-stimulating factor(rhG-CSF), or granulocyte-macrophage colony-stimulating factor(rhGM-CSF) on the expression of Fc R II, Fc R III, CD11c and CD18 and respiratory burst function of granulocytes. METHODS: Peripheral blood was collected from six healthy adults. The isolated granulocytes were stimulated with rhG-CSF, 250mg/mL, rhGM-CSF, 100ng/mL or both of them for 1, 3, 9, 24, and 48 hours. Using flow cytometry, we compared the expression of the Fc R II, Fc R III, CD11c and CD18 of granulocytes before and after stimulation. We also the studied respiratory burst of granulocytes with chemiluminescence assay. RESULTS: Fc R II and CD18 expression were not changed significantly after stimulation. Fc R III expression was decreased significantly after stimulation with rhG-CSF, rhGM-CSF, or both of them. CD11c expression was increased initially but was found to decrease significantly after 9 hours of stimulation. Granulocytes treated with rhG-CSF, rhGM-CSF, or both displayed enhanced respiratory burst. Our results suggest that exposure of granulocyte to growth factor results in granulocyte activation. CONCLUSION: We have shown that rhG-CSF and rhGM-CSF resulted in an activation of peripheral blood granulocytes immunophenotypically and functionally.

Antibody Levels and Opsonophagocytic Function to S. pneumoniae 19F Capsular Polysaccharide

PURPOSE: The increasing prominence of pneumococcal infections and of antimicrobial resistance has emphasized the need for a effective vaccine to reduce the risk of pneumococcal disease in childhood. To study the antibody response of pneumococcal vaccine, we compared the concentration and opsonophagocytic capacity of antibodies to the capsular polysaccharide of S. pneumoniae serotype 19F in sera from healthy adults after immunization with 23-valent pneumococcal polysaccharide(PS) vaccine. METHODS: Thirty healthy adults were immunized with pneumococcal 23-valent PS vaccine. One month after vaccination, the amounts of total, IgG, IgG1, and IgG2 antibody were determined by enzyme-linked immunosorbent assay(ELISA). To investigate the function of anti-19F antibodies, the opsonization titer was determined by opsonophagocytic assay. RESULTS: The mean antibody levels of total immunoglobulin and IgG specific to 19F serotype were 21.9mg/mL and 16.2mg/mL, respectively. Most of the IgG anti-PS antibodies were IgG2 isotype. The mean value of opsonization titer was 229.1. The opsonization titer against the 19F serotype was strongly correlated with total immunoglobulin and IgG anti-19F antibody concentration. And IgG2 concentration was correlated more with opsonization titer than with IgG1. CONCLUSION: Antibody level may be of general use in predicting vaccine-induced protection among adults for 19F pneumococcus. This study should be extended to other serotypes and to assess the antibody response to new protein conjugate pneumococcal vaccine in young children.